Phospholipid flippases translocate phospholipids from the exoplasmic to the cytoplasmic leaflet of cell membranes to generate and maintain phospholipid asymmetry. The genome of budding yeast encodes four heteromeric flippases (Drs2p, Dnf1p, Dnf2p, and Dnf3p), which associate with the Cdc50 family noncatalytic subunit, and one monomeric flippase Neo1p. Flippases have been implicated in the formation of transport vesicles, but the underlying mechanisms are largely unknown. We show here that overexpression of the phosphatidylserine synthase gene CHO1 suppresses defects in the endocytic recycling pathway in flippase mutants. This suppression seems to be mediated by increased cellular phosphatidylserine. Two models can be envisioned for the suppression mechanism: (i) phosphatidylserine in the cytoplasmic leaflet recruits proteins for vesicle formation with its negative charge, and (ii) phosphatidylserine flipping to the cytoplasmic leaflet induces membrane curvature that supports vesicle formation. In a mutant depleted for flippases, a phosphatidylserine probe GFP-Lact-C2 was still localized to endosomal membranes, suggesting that the mere presence of phosphatidylserine in the cytoplasmic leaflet is not enough for vesicle formation. The CHO1 overexpression did not suppress the growth defect in a mutant depleted or mutated for all flippases, suggesting that the suppression was dependent on flippase-mediated phospholipid flipping. Endocytic recycling was not blocked in a mutant lacking phosphatidylserine or depleted in phosphatidylethanolamine, suggesting that a specific phospholipid is not required for vesicle formation. These results suggest that flippase-dependent vesicle formation is mediated by phospholipid flipping, not by flipped phospholipids.

Budding yeast mutants in assembly of actin cables, which are thought to be the only actin structures essential for budding, still could form a small bud. Mutations in actin patch endocytic machineries/endocytic recycling factors inhibited this budding, suggesting a mechanism that promotes polarized growth by local recycling of endocytic vesicles.

Vitamin A and its derivatives, particularly retinol, slow both chronological and photo-induced aging processes. Retinol reduces the appearance of wrinkles and the decrease of the skin firmness and elasticity by protecting against collagen and elastin fiber changes. In addition to retinol's rejuvenating skin benefits through direct transcriptional activation, recent studies also suggest an epigenetic regulation through micro-RNAs (miRNAs) modulation. We investigated how retinol supports the stimulation of type I collagen and elastin through proteomic, transcriptomic and epigenetic miRNA-expression changes in human skin fibroblasts. Five miRNAs, previously described as targeting collagen or elastin gene expression, were particularly studied after validation of their expression in normal human fibroblasts by miRNA sequencing analysis. Retinol treatment was applied on monolayered cultures of dermal fibroblasts and the levels of the 5 selected miRNAs were quantified by q-RT-PCR. We observed that retinol was able to reduce the expression of the 5 studied collagen-or elastin-inhibiting miRNAs. After retinol treatment, miR-181b (related to elastin protein) and Let7b (related to collagen protein) were respectively decreased by 39% and 37% In addition, miR-29b, miR-195 and miR-497, which regulate both collagen and elastin proteins, were respectively decreased by 24%, 49% and 42% after retinol treatment. In addition, retinol simultaneously stimulated both Elastin and collagen type I proteins by 38% and 296% respectively. These experiments efficiently characterize the epigenetic activity of retinol through inhibition of specific miRNAs. This may contribute to increase the knowledge on retinol mode of action related to rejuvenating skin benefits. 168ARP-T1-associated Bazex-Dupre ´-Christol Syndrome is an inherited basal cell cancer with ciliary defects characteristic of ciliopathies